Industry 4.0 is driving the revolution of manufacturing processes by combining innovative technologies and new interaction paradigms among systems and operators. In particular, the layout, tasks and work sequences of assembly lines are designed according to several transdisciplinary Design Principles (DPs), such as process efficiency, product quality, ergonomics, safety and operators’ workload. A large variety of simulation software can be employed for evaluations. However, the related ability to assess multidisciplinary factors must be evaluated. The paper aims to provide a framework for guiding the assessment of simulation software in the context of Industry 4.0 assembly lines. Process requirements are first analyzed and mapped to select DPs, prioritized according to design goals by an analytical hierarchy process. Then, suitable simulation software is determined accordingly, and the virtual model is realized. Finally, the possibility of the software to provide meaningful elaborations for the selected DPs is assessed. The framework has been tested on a prototypal Industry 4.0 assembly line composed of automated logistic systems, cobots and systems to guide the execution of tasks. The line has been modeled in Siemens Process Simulate, analyzing the completeness and appropriateness of the functionalities of this software according to the defined DPs.